Assay for assessing conformational stability of membrane protein

ABSTRACT

The invention provides an assay for assessing the conformational stability of a membrane protein, comprising: (a) providing a sample comprising a first population and a second population of a membrane protein; wherein the membrane protein in the first population is labelled with a donor label and the membrane protein in the second population is labelled with an acceptor label, or the membrane protein in the first population is labelled with an acceptor label and the membrane protein in the second population is labelled with a donor label, (b) exposing the first and second populations of the membrane protein to a stability modulating agent and/or condition, (c) and assessing aggregation between membrane proteins of the first and second populations by activating the donor label to permit a distance-dependent interaction with the acceptor label, which interaction produces a detectable signal.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. § 371 ofinternational application PCT/GB2013/051464, filed May 31, 2013,entitled “Assays,” which claims the benefit under 35 U.S.C. § 119(e) ofU.S. Provisional Application Ser. No. 61/654,265, entitled “Assays,”filed on Jun. 1, 2012, each of which is herein incorporated by referencein its entirety.

The present invention relates to assays for assessing the stability of amembrane protein, especially a G protein-coupled receptor (GPCR).

Over the past 20 years the rate of determination of membrane proteinstructures has gradually increased, but most success has been incrystallising membrane proteins from bacteria rather than fromeukaryotes. Bacterial membrane proteins have been easier to overexpressusing standard techniques in Escherichia coli than eukaryotic membraneproteins [1] and the bacterial proteins are sometimes far more stable indetergent, detergent-stability being an essential prerequisite topurification and crystallisation. Apart from the difficulties inoverexpressing eukaryotic membrane proteins, they often have poorstability in detergent solutions, which severely restricts the range ofcrystallisation conditions that can be explored without their immediatedenaturation or precipitation. Ideally, membrane proteins should bestable for many days in any given detergent solution, but the detergentsthat are best suited to growing diffraction-quality crystals tend to bethe most de-stabilising detergents i.e. those with short aliphaticchains and small or charged head groups. It is also the structures ofhuman membrane proteins that we would like to solve, because these arerequired to help the development of therapeutic agents by thepharmaceutical industry. There is thus an overwhelming need to develop ageneric strategy that will allow the production of detergent-stableeukaryotic integral membrane proteins for crystallisation and structuredetermination and potentially for other purposes such as drug screening,bioassay and biosensor applications.

GPCRs constitute a very large family of proteins that control manyphysiological processes and are the targets of many effective drugs.Accordingly, they are of considerable pharmacological importance. A listof GPCRs is given in Foord et al (2005) Pharmacol Rev. 57, 279-288,which is incorporated herein by reference. GPCRs are generally unstablewhen isolated, and despite considerable efforts, few crystal structuresexist.

GPCRs are druggable targets, and reference is made particularly toOverington et al (2006) Nature Rev. Drug Discovery 5, 993-996 whichindicates that over a quarter of present drugs have a GPCR as a target.

GPCRs are thought to exist in multiple distinct conformations which areassociated with different pharmacological classes of ligand such asagonists and antagonists, and to cycle between these conformations inorder to function (Kenakin T. (1997) Ann N Y Acad Sci 812, 116-125).

We have realised that there are two serious problems associated withtrying to crystallise GPCRs, namely their lack of stability (eg indetergent) and the fact that they exist in multiple conformations. Inorder to function, GPCRs have evolved to cycle through at least twodistinct conformations, the agonist-bound form and the antagonist-boundform, and changes between these two conformations can occurspontaneously in the absence of ligand. It is thus likely that anypurified receptors populate a mixture of conformations. Just addingligands to GPCRs during crystallisation trials has not resulted in theirstructure determination.

We found that stabilised receptors (StaRs) can be generated through asystematic mutagenesis scan throughout the whole receptor combined witha thermostability assay based on ligand binding. The method of StaRgeneration results in the stabilisation of a specific conformation ofreceptor. A detailed description of the method can be found elsewhere[2, 3, 4] and in WO 2008/114020 and WO 2009/071914. Briefly, in order tostabilise the receptors in a particular conformation, the solubilisedreceptor is incubated with an excess amount of either the agonist orantagonist, depending on the desired conformation. The receptor/ligandcomplex is then heated at various temperatures for a certain length oftime. Following the heating, the excess unbound ligand is separated fromreceptor/ligand complexes. The percentage of ligand binding at any giventemperature is used as readout for the amount of folded receptorremaining following heating. Plotting this data against temperatureproduces a thermal decay curve and the Tm value is defined as thetemperature at which 50% of receptor activity is retained. This processrelies on the availability of a good ligand with high affinity andfavourable properties in detergent. However, in some cases such a ligandis not available and so an assay that circumvents the need for a ligand,i.e. where ligand binding activity is not used to measure receptoractivity, is highly desirable.

Historically, structural changes of proteins during heating have beenstudied by various analytical techniques, such as sedimentationvelocity, differential scanning calorimetry, dynamic light scattering,circular dichroism spectroscopy, UV/VIS spectroscopy, electrophoresis,fluorescence and NMR. However, these techniques often require largeamounts of protein, are difficult to adapt to high-throughput screening,and often suffer from poor signal-to-noise ratios due to high backgroundnoise from detergents.

Another method for measuring the thermal stability of membrane proteinswithout the use of a ligand has been described by Alexandrov et al [5].This relies on the accessibility of native cysteine residues to covalentmodification as a readout for the unfolding process. Protein unfoldingresults in the exposure of embedded cysteines which makes them prone tomodification with a reactive fluorescent probe. In the known assay theauthors use the sulfhydryl-specific fluorochromeN-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM)because it exhibits little fluorescence in its unbound form. A majordisadvantage of this assay, however, is that it is unsuitable for highthroughput applications. It requires quite large quantities of a highlypure membrane protein preparation because the detection system is notspecific for the membrane protein.

Knepp et al (Biochemistry 50(4): 502-11, 2011) describe the use ofFluorescence Resonance Energy Transfer (FRET) [6] to assess membraneprotein stability. FRET relies on the distance-dependent transfer ofenergy from a donor fluorophore to an acceptor fluorophore. Importantly,donor and acceptor fluorophores must have well separated emissionspectra, but the emission spectrum of the donor must overlap with theexcitation spectrum of the acceptor fluorophore. In Knepp et al'smethod, binding of a monoclonal antibody labelled with a FRET acceptoris used as a marker of GPCR denaturation. A FRET signal is generatedwhen the FRET acceptor is in the proximity of the FRET donor attached tothe C-terminus of the GPCR. A significant limitation of this method,however, is that it requires the availability of suitable antibodiesspecific for the membrane protein in question. Also, the method of Kneppet al is unlikely to identify stabilising mutations that happen to be inthe antibody binding site since these mutations will reduce or abolishthe antibody binding. Additionally, mutations in the antibody bindingsite may increase the affinity of the antibody for the receptor withoutnecessarily increasing conformational stability, thereby leading tofalse positives. Another disadvantage of Knepp et al's method is thatthe antibody may have a specific effect on the pharmacology of the GPCR.For example, the antibody may be a particular conformation of the GPCRthat is not desired.

Hence, there remains a great need for a simple effective assay which canbe used to measure the stability of membrane proteins.

Protein aggregation upon unfolding is a general feature of all proteinsthat occurs to varying degrees in different proteins. It is acceptedthat the overall structural stability of a protein is inverselyproportional to the levels of aggregation. Membrane proteins are knownto exhibit high levels of aggregation after solubilisation and more soafter denaturation.

There are various methods available for measuring aggregation, but notfor measuring membrane protein aggregation following denaturation.

For example, Pollitt et al (Neuron 40: 685-694, 2003) and U.S. Pat. No.7,803,559 describe a cell-based assay to measure intracellularpolyglutamine protein aggregation using FRET, and discuss its use inscreening for aggregation regulators that may have therapeuticapplication in neurodegenerative diseases. Also, Roberti et al (NatureMethods 4(4): 345, 2007) and Rajan et al (PNAS 98(23): 13060-13065)describe FRET based methods for assessing the aggregation of proteinsthat are known to form aggregates as part of a disease pathology.

Protein aggregation may also be measured by the analytical techniquesmentioned above. However, all of these methods demand highly purifiedprotein

Despite the above advances, methods for measuring the aggregation ofmembrane proteins are not widely available. This is mainly because thesemethods require large amounts of highly purified protein and generatinghighly purified membrane protein is challenging.

The present inventors have now identified methods for directly assessingaggregation of membrane proteins. Such methods allow one to identify theconditions and mutations that can minimise overall aggregation of theproteins. Compared to methods that indirectly measure aggregation suchas that of Knepp et al, the present methods are believed to identifymore mutations and conditions that can minimise aggregation of proteins.This is because some mutations and conditions may reduce proteinaggregation without changing the structure of the protein in a way thatwould lead to a change in ligand binding or antibody binding, as isrelied on as a read out for aggregation in the indirect methods.

According to a first aspect of the invention there is provided an assayfor assessing the conformational stability of a membrane protein,comprising:

(a) providing a sample comprising a first population and a secondpopulation of a membrane protein; wherein the membrane protein in thefirst population is labelled with a donor label and the membrane proteinin the second population is labelled with an acceptor label, or themembrane protein in the first population is labelled with an acceptorlabel and the membrane protein in the second population is labelled witha donor label,

(b) exposing the first and second populations of the membrane protein toa stability modulating agent and/or condition,

(c) and assessing aggregation between membrane proteins of the first andsecond populations by activating the donor label to permit adistance-dependent interaction with the acceptor label, whichinteraction produces a detectable signal.

In this assay, aggregation of the membrane proteins from the first andsecond populations is detected by virtue of the donor labels andacceptor labels coming into proximity with each other, and therebygenerating a detectable signal, when the membrane proteins aggregate. Ifthe membrane proteins are stable, there is less aggregation uponexposure to a destabilising agent and/or condition and so less signal,whereas if the membrane proteins are unstable, there is more aggregationupon exposure to a destabilising agent and/or condition and so moresignal.

By “conformational stability” we include the meaning of the stability ofa particular conformation of a membrane protein (eg GPCR). The stabilityof a particular conformation refers to how well that particularconformation can retain its structure when exposed to a denaturant ordenaturing conditions. Thus, a membrane protein with high conformationalstability will have an extended lifetime of a particular conformationcompared to the lifetime of a particular conformation of a membraneprotein with low conformational stability. Examples ofdenaturants/denaturing conditions include heat, detergent, a chaotropicagent and an extreme of pH, as described further below. As is well knownin the art, such denaturants or denaturing conditions can affectsecondary and tertiary structures of a protein but not the primarysequence.

By “membrane protein” we mean a protein that is attached to orassociated with a membrane of a cell or organelle. Preferably, themembrane protein is an integral membrane protein that is permanentlyintegrated into the membrane and can only be removed using detergents,non-polar solvents or denaturing agents that physically disrupt thelipid bilayer.

Most preferably, the membrane protein is a GPCR.

Suitable GPCRs for use in the practice of the invention include, but arenot limited to chemokine receptor, (eg CCR5), β-adrenergic receptor,adenosine receptor (eg A_(2a) receptor), and neurotensin receptor (NTR).Other suitable GPCRs are well known in the art and include those listedin Hopkins & Groom supra. In addition, the International Union ofPharmacology produce a list of GPCRs (Foord et al (2005) Pharmacol. Rev.57, 279-288, incorporated herein by reference and this list isperiodically updated athttp://www.iuphar-db.org/GPCR/ReceptorFamiliesForward). It will be notedthat GPCRs are divided into different classes, principally based ontheir amino acid sequence similarities. They are also divided intofamilies by reference to the natural ligands to which they bind. AllGPCRs are included in the scope of the invention.

Thus, the GPCR may be any of an adenosine receptor, a β-adrenergicreceptor, a neurotensin receptor, a muscarinic acid receptor, a5-hydroxytryptamine receptor, an adrenoceptor, an anaphylatoxinreceptor, an angiotensin receptor, an apelin receptor, a bombesinreceptor, a bradykinin receptor, a cannabinoid receptor, a chemokinereceptor, a cholecystokinin receptor, a dopamine receptor, an endothelinreceptor a free fatty acid receptor, a bile acid receptor, a galaninreceptor, a motilin receptor, a ghrelin receptor, a glycoprotein hormonereceptor, a GnRH receptor, a histamine receptor, a KiSS1-derived peptidereceptor, a leukotriene and lipoxin receptor, a lysophospholipidreceptor, a melanin-concentrating hormone receptor, a melanocortinreceptor, a melatonin receptor, a neuromedin U receptor, a neuropeptidereceptor, a N-formylpeptide family receptor, a nicotinic acid receptor,an opiod receptor, an opsin-like receptor, an orexin receptor, a P2Yreceptor, a peptide P518 receptor, a platelet-activating factorreceptor, a prokineticin receptor, a prolactin-releasing peptidereceptor, a prostanoid receptor, a protease-activated receptor, arelaxin receptor, a somatostatin receptor, a SPC/LPC receptor, atachykinin receptor, a trace amino receptor, a thryotropin-releasinghormone receptor, an urotensin receptor, a vasopressin/oxytocinreceptor, an orphan GPCR, a calcitonin receptor, a corticotropinreleasing factor receptor, a glucagon receptor, a parathyroid receptor,a VIP/PACAP receptor, a LNB7TM receptor, a GABA receptor, a metabotropicglutamate receptor, and a calcium sensor receptor (see Table 1 of Foordet al (2005) Pharmacol. Rev. 57, 279-288, incorporated herein byreference).

The GPCR may also be selected from any of the GPCRs listed in Table Ahereinafter.

Also, other membrane proteins for which the methods of the invention mayusefully be employed to measure stability include the orphan receptorslisted in Table B hereinafter.

The amino acid sequences (and the nucleotide sequences of the cDNAswhich encode them) of many membrane proteins (eg GPCRs are readilyavailable, for example by reference to GenBank. In particular, Foord etal supra gives the human gene symbols and human, mouse and rat gene IDsfrom Entrez Gene (accessible online at ncbi.nlm.nih.gov/entrez). Itshould be noted, also, that because the sequence of the human genome issubstantially complete, the amino acid sequences of human membraneproteins (eg GPCRs) can be deduced therefrom.

Although the membrane protein (eg GPCR) may be derived from any source,it is particularly preferred if it is from a eukaryotic source. It isparticularly preferred if it is derived from a vertebrate source such asa mammal or a bird. It is particularly preferred if the membrane protein(eg GPCR) is derived from rat, mouse, rabbit or dog or non-human primateor man, or from chicken or turkey. For the avoidance of doubt, weinclude within the meaning of “derived from” that a cDNA or gene wasoriginally obtained using genetic material from the source, but that theprotein may be expressed in any host cell (eg prokaryotic or eukaryotichost cell) subsequently. Thus, it will be plain that a eukaryoticmembrane protein (eg GPCR) (such as an avian or mammalian membraneprotein) may be expressed in a prokaryotic host cell, such as E. coli,but be considered to be avian- or mammalian-derived, as the case may be.

In some instances, the membrane protein may be composed of more than onedifferent subunit. For example, the calcitonin gene-related peptidereceptor requires the binding of a single transmembrane helix protein(RAMP1) to acquire its physiological ligand binding characteristics.Also, effector, accessory, auxiliary or GPCR-interacting proteins whichcombine with the GPCR to form or modulate a functional complex are wellknown in the art and include, for example, receptor kinases, G-proteinsand arrestins (Bockaert et al (2004) Curr Opinion Drug Discov and Dev 7,649-657).

The membrane proteins (eg GPCRs) may be prepared by any suitable method.Conveniently, the membrane protein is encoded by a suitable nucleic acidmolecule and expressed in a suitable host cell. Suitable nucleic acidmolecules encoding the membrane protein (eg GPCR) may be made usingstandard cloning techniques, site-directed mutagenesis and PCR as iswell known in the art. Suitable expression systems include constitutiveor inducible expression systems in bacteria or yeasts, virus expressionsystems such as baculovirus, semliki forest virus and lentiviruses, ortransient transfection in insect or mammalian cells. Suitable host cellsinclude E. coli, Lactococcus lactis, Saccharomyces cerevisiae,Schizosaccharomyces pombe, Pichia pastoris, Spodoptera frugiperda andTrichoplusiani cells. Suitable animal host cells include HEK 293, COS,S2, CHO, NSO, DT40 and so on. It is known that some membrane proteins(eg GPCRs) require specific lipids (eg cholesterol) to function. In thatcase, it is desirable to select a host cell which contains the lipid.Additionally or alternatively the lipid may be added during isolationand purification of the membrane protein. It may also be desirable toadd a ligand of the membrane protein (eg GPCR) as explained furtherbelow.

Molecular biological methods for cloning and engineering genes andcDNAs, for mutating DNA, and for expressing polypeptides frompolynucleotides in host cells are well known in the art, as exemplifiedin “Molecular cloning, a laboratory manual”, third edition, Sambrook, J.& Russell, D. W. (eds), Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., incorporated herein by reference.

The sample comprising the first population and second population of amembrane protein may be any suitable sample that contains the twopopulations. By “population” we include a plurality of the same specifictype of membrane protein, as opposed to a mixture of different proteins.For example, the population may comprise at least 2, 5, 10, 50, 100,200, 500, 1000, 5000, 10000, 100000, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹,10¹², 10¹³ or 10¹⁴ protein molecules, or more. It will be appreciatedthat the precise number of protein molecules may depend on theexpression level of the particular membrane protein and this may differfrom protein to protein.

It is preferred if the populations of membrane protein (eg GPCR) areprovided in a suitable solubilised form in which the membrane proteinsmaintain structural integrity and are in a functional form (eg are ableto bind ligand). An appropriate solubilising system, such as a suitabledetergent (or other amphipathic agent) and buffer system is used, whichmay be chosen by the person skilled in the art to be effective for theparticular protein. Typical detergents which may be used include, forexample, dodecylmaltoside (DDM) or CHAPS or octylglucoside (OG) or manyothers. In an embodiment, the sample comprises one or more detergentsselected from DDM; C₁₁-, C₁₀-, C₉- or C₈-maltoside or glucoside; LDAO;and SDS. It may be convenient to include other compounds such ascholesterol hemisuccinate or cholesterol itself or heptane-1,2,3-triol.The presence of glycerol or proline or betaine may be useful. It isimportant that the membrane protein (eg GPCR), once solubilised from themembrane in which it resides, must be sufficiently stable to be assayed.For some membrane proteins (eg GPCRs), DDM will be sufficient, butglycerol or other polyols may be added to increase stability for assaypurposes, if desired. Further stability for assay purposes may beachieved, for example, by solubilising in a mixture of DDM, CHAPS andcholesterol hemisuccinate, optionally in the presence of glycerol. Forparticularly unstable membrane proteins (eg GPCRs), it may be desirableto solubilise them using digitonin or amphipols or other polymers whichcan solubilise membrane proteins directly from the membrane, in theabsence of traditional detergents and maintain stability typically byallowing a significant number of lipids to remain associated with themembrane protein. Nanodiscs may also be used for solubilising extremelyunstable membrane proteins in a functional form.

Typically, the membrane protein is provided in a crude extract (eg ofthe membrane fraction from the host cell in which it has been expressed,such as E. coli or HEK293T cells). It may be provided in a form in whichthe membrane protein comprises at least 75%, or at least 80% or 85% or90% or 95% or 98% or 99% of the protein present in the sample.Alternatively, the membrane protein may be provided in a semi-purifiedform as described in Example 1 wherein the membrane protein of interestis not the most abundant species in the sample. Thus, the membraneprotein may be provided in a sample where only 5-50% of the totalprotein in the sample is the membrane protein (eg at least 5% or 10% or15% or 20% or 25% or 30% or 35% or 40% or 45% of the protein present inthe sample). However, for the assay of the first aspect of theinvention, some purification of the membrane protein following itsexpression is generally required and so the crude lysate cannot normallybe used. Any suitable protein purification method may be used as isstandard practice in the art. Of course, it is typically solubilised asdiscussed above, and so the membrane protein is usually associated withdetergent molecules and/or lipid molecules.

Conveniently, the first and second populations of the membrane proteinare expressed and labelled separately. The populations may also besolubilised and/or purified separately as discussed above. The first andsecond populations of the membrane protein are then mixed together toobtain a sample. Preferably, the first population and second populationof the membrane protein are present in the sample in a 1:1 molar ratio.However, it is appreciated that for small scale protein purifications,it may be difficult to measure the concentrations of proteins and so a1:1 molar ratio is typically approximated by a 1:1 volume ratio. Theapproximation is believed to be close since size differences between thetwo populations are expected to be small and the purification efficiencyis thought to be the same. Conveniently, a 1:1 volume ratio is achievedby solubilising an equal number of cells from a first population ofcells expressing the membrane protein and a second population of cellsexpressing the membrane protein, purifying the membrane proteins fromeach population identically and eluting the purified protein in the samevolume. The eluates are then mixed in equal volumes, so for example 250μl of the first population is mixed with 250 μl of the second populationand so on.

It will be appreciated that one of the populations of membrane proteinsin the sample will be labelled with a donor label (not an acceptorlabel) and the other of the populations of membrane proteins will belabelled with an acceptor label (not a donor label). By ‘donor label’and ‘acceptor label’ we include the meaning of any pairs of labels,where activation of the donor label permits a distance-dependentinteraction with the acceptor label, which interaction produces adetectable signal. In this way, the detectable signal generated byinteraction of donor and acceptor pairs can be used as a readout of thelevel of aggregation between membrane proteins from the first and secondpopulations, and therefore conformational stability.

Conveniently, the interaction between the donor label and acceptor labelinvolves the transfer of energy from the donor label to the acceptorlabel. For example, the term resonance energy transfer relates to amethod where a donor label is in a close vicinity to an acceptor label.This generates an energy flow from the donor to the acceptor leading toa detection scheme where a signal is monitored through the donor oracceptor. Such a method is well known for example in a luminescenceresonance energy transfer system where the donor dye can be a down or upconverting label. The donor is excited and as a consequence of theproximity principle the acceptor label is excited by the donor compoundand a signal is detected at the emission wavelength of the acceptorcompound. There are a number of resonance energy transfer methods suchas fluorescence, phosphorescence, time-resolved fluorescence,bioluminescence and luminescence resonance energy transfer. Theresonance energy transfer can be realised as a signal generating methodor a method where the signal is quenched. In the case of quenching, anyelement can be used to quench signal such as a dye or metal. In such acase, typically the emission wavelength of the donor molecule isdetected. Typical metal chelating or complexing agents or ligands usedin time-resolved fluorometry are 3-(2-thienoyl)-1,1,1-trifluoroacetone,3-benzoyl-1,1,1-trifluoroacetone, coproporphyrins, porphyrins,3-naphthoyl-1,1,1-trifluoroacetone,2,2-dimethyl-4-perfluorobutyoyl-3-butanone, 2,2′-dipyridyl,phenanthroline, salicylic acid, phenanthroline carboxylic acid,aminophenanthroline, diphenylphenantroline, dimethylphenanthroline,bipyridylcarboxylic acid, aza crown ethers, trioctylphosphine oxide, azacryptands, dibenzoylmethane, dinaphtoylmethane, dibiphenoylmethane,benzoylacetonato, phenylazodibenzoylmethane, dithienylpropanedione,4,4′-bis(N,N-dimethylamino)benzophenone,tris(6,6,7,7,8,8,8-heptafluoro-2,2-dimethyloctane-3,5-dione,(alkyloxyphenyl)pyridine-2,6-dicarboxylic acid and their derivatives.Metal ions can be, for example, lanthanide or ruthenium ions. (Selvin P.Nature Struc. Biol. 2000:7; 730; Forster T. Discuss. Faraday Soc.1959:27; 7; Latva M. Academic Dissertation, University of Turku,Finland, 1997; Mukkala V.-M. Academic Dissertation, University of Turku,Finland, 1993; Wohrle D. and Pomogailo A. D. Metal Complexes and Metalsin Macromolecules, John Wiley & Sons, 2003).

In a preferred embodiment, the interaction between the donor andacceptor labels involves fluorescence energy transfer (FRET), aphenomenon whereby when donor and acceptor fluorophores are in closeproximity, the fluorescence energy of the donor fluorophore transfers tothe acceptor fluorophore, and luminescence of the acceptor fluorophoreis observed. Any suitable pair of donor and acceptor fluorophores may beused as the donor and acceptor labels.

A particularly preferred donor fluorophore is a lanthanide, such aseuropium, terbium, samarium or dysprosium. Lanthanides have longlifetimes that enable time-resolved measurement of acceptor emission.This is important because it significantly decreases the effect ofautofluorescence from biological material and possible direct acceptorexcitation. Also, the emission peaks of lanthanides are well separated,narrow and exhibit long Stokes' shifts (the distance between the maximumabsorption and emission wavelengths).

The acceptor fluorophore may be any fluorophore with an excitation wavelength that overlaps with one of the emission peaks of the donor. Forthe emission peaks of the lanthanide chelates see Table 1, the dominantpeak is shown in bold. For example, with Terbium donor, any fluorophorewith the excitation wave length around 490 nm, 545 nm or 620 nm could beused. Hence, a fluorophore such as Fluorescein, Rhodamine, Alexa Fluora488, Dylight 488, d2, Cy3, BODIPY FL, BODIPY 630/650-X, red-shiftedvariants of green fluorescent protein (GFP), typified by EGFP andGFP-S65T, may be used in combination with Terbium.

TABLE 1 Eu3+ Dy3+ Tb3+ Sm3+ 580 nm 483 nm 490 nm 560 nm 590 nm 575 nm545 nm 598 nm 613 nm 660 nm 590 nm 643 nm 650 nm 620 nm 710 nm 690 nm650 nm 710 nm

In an alternative embodiment, the interaction between the donor labeland the acceptor label involves bioluminescence energy transfer (BRET).This differs from FRET in that the donor fluorophore of the FRETtechnique is replaced by a bioluminescent protein such as luciferase. Inthe presence of a substrate, bioluminescence from the bioluminescentprotein excites the acceptor label by the same Förster resonance energytransfer mechanism as for FRET.

A criteria which should be considered in determining suitable pairingsfor BRET is the relative emission/fluorescence spectrum of the acceptorlabel compared to that of the bioluminescent protein. The emissionspectrum of the bioluminescent protein should overlap with theabsorbance spectrum of the acceptor protein such that the light energyfrom the bioluminescent protein luminescence emission is at a wavelengththat is able to excite the acceptor label and thereby promote acceptorlabel fluorescence when the two molecules are in a proper proximity andorientation with respect to one another. Two common implementations ofBRET that may be used comprise Renilla luciferase (RLuc) with eithercoelenterazine h (BRET¹; λ_(em)=^(˜)475 nm) or coelenterazine 400a(Clz400a) substrate (BRET²; λ_(em)=^(˜)395 nm) as the donor systemcoupled to either of the GFP mutants, YFP (BRET¹; λ_(em)=^(˜)530 nm) orGFP² (BRET²; λ_(em)=^(˜)510 nm). However, any suitable pairs of BRETdonor and acceptor labels may be used that are known in the art, and asreviewed for example in Xia and Rao (Curr Opin Biol 20: 1-8, 2009).

Renilla luciferase/EGFP pairing has been compared to Renillaluciferase/EYEF pairing based on observable emission spectral peaks (Xu,1999; Wang, et al (1997) in Bioluminescence and Chemiluminescence:Molecular Reporting with Photons, eds. Hastings et al (Wiley, New York),pp. 419-422). To study potential pairing, protein fusions are preparedcontaining the selected bioluminescent protein and acceptor molecule andare tested, in the presence of an appropriate substrate.

Preferably, the donor and acceptor labels are attached to the membraneprotein at either the N-terminus or the C-terminus of the membraneprotein. Labels attached to the termini are likely to have minimaleffects on the structure and activity of the membrane protein. However,in principle, the labels can be placed in other regions of the membraneprotein provided that they do not prevent folding of the membraneprotein and/or interfere with its activity. Techniques to assess proteinfolding and activity to enable the skilled person to assess this arestandard practice in the art.

It is preferred if the membrane proteins of the first and secondpopulations are labelled with the donor and acceptor labels such thatthe labels are covalently attached to the membrane protein, for exampleat the N- or C-terminus of the membrane protein. For example, themembrane protein in the first population may be covalently attached to adonor label and the membrane protein in the second population may becovalently attached to an acceptor label, or the membrane protein in thefirst population may be covalently attached to an acceptor label and themembrane protein in the second population may be covalently attached toa donor label.

Techniques to covalently attach donor and acceptor labels to proteinsare well known in the art, and any suitable method, including chemicalconjugation, may be used.

For example, the Tag-Lite™ system, marketed by Cisbio may be used [7, 8,9]. Here, the membrane protein of interest is cloned into an expressionvector upstream or downstream of a SNAP or a CLIP tag. The SNAP and CLIPtags are related proteins that are modified forms of the mammalianO6-alkylguanine-DNA-alkyltransferase (AGT). The SNAP-tag and CLIP-tagsubstrates are derivates of benzyl purines and benzyl pyrimidines, wherethe benzyl group is attached to a functional group such as afluorophore. During the labelling reaction, the modified benzyl groupreacts with a free cysteine in AGT and the functional group is attachedcovalently. The expression of the membrane protein from this plasmidresults in the production of a membrane protein having the SNAP or CLIPtag at the N-terminus or C-terminus. Membrane proteins can then belabeled with lanthanides (such as Terbium or Europium) that act as donorfluorophores. Thus, the membrane protein may be labelled with a donor oracceptor label (eg a donor or acceptor fluorophore) via a terminus tag,eg a C-terminal or N-terminal tag, such as the SNAP and CLIP tags usingthe Tag-Lite™ system. It will appreciated that for correct labelling ofthe C-terminus, the SNAP-tag or CLIP-tag substrate attached to thefunctional group (eg fluorophore) must be capable of crossing the cellmembrane.

For donor and acceptor labels that are polypeptides (eg GFP), it will beunderstood that the labels may be covalently attached to the membraneprotein by forming a fusion polypeptide. Preparation of fusionpolypeptides is routine practice in the art and may involve chemicalconjugation or may involve recombinant technology where the labelledmembrane protein is expressed by a single polynucleotide.

In an alternative embodiment, the donor and acceptor labels may beattached to the membrane proteins non-covalently provided that thenon-covalent interaction is strong enough to withstand the stabilitymodulating agent and/or condition in step (b). Such non-covalentbindings may include immunological bindings or bindings such as viabiotin/avidin or streptavidin. Whether the non-covalent interaction issufficiently strong can be determined by exposing the labelled membraneprotein to the stability modulating agent and/or condition and using anappropriate analytical technique to assess whether the label remainsbound to the membrane protein following exposure.

It will be appreciated that the donor label or acceptor label may beattached to the membrane directly or indirectly. By ‘attached to themembrane protein directly’, we include the meaning of the label beingeither covalently or non-covalently attached to the membrane protein. By‘attached to the membrane protein indirectly’, we include the meaning ofthe label being either covalently or non-covalently attached to afurther moiety which, in turn, is covalently or non-covalently attachedto the membrane protein.

Following exposure to the stability modulating agent and/or condition,the donor label is activated to permit a distance-dependent interactionwith the acceptor label, and the level of aggregation assessed. Byactivating the donor label we include the meaning of initiating aprocess whereby there is a distance-dependent interaction between thedonor and acceptor labels. The process may be one that results in theemission by the donor of a photon, or transfer of energy throughresonance energy transfer or some other method. Examples of donoractivators include a photon (as in the case of a FRET donor) or asubstrate of a bioluminescent protein (as in the case of a BRET donor).Thus, activating the donor label may comprise irradiating the donorlabel or exposing the donor label to a substance, so as to lead to adistance dependent interaction with the acceptor label.

In an embodiment, it may be desirable to also perform steps (a) and (c)of the method without performing step (b). This would provide anindication of background aggregation when the membrane protein is notexposed to a stability modulating agent and/or condition that can becompared to the level of aggregation when the membrane protein isexposed to a stability modulating agent and/or condition. In otherwords, the assay may be preceded by a step wherein the aggregationbetween membrane proteins of the first and second populations isassessed without exposure to a stability modulating agent and/orcondition.

A second aspect of the invention provides an assay for assessing theconformational stability of a membrane protein, comprising:

(a) providing a sample comprising a membrane protein population,

(b) exposing the membrane protein population to a stability modulatingagent and/or condition,

(c) labelling one of the N-terminus or C-terminus of the membraneprotein with a donor label and the other of the N-terminus or C-terminusof the membrane protein with an acceptor label,

(d) and assessing aggregation of the membrane proteins in the populationby activating the donor label to permit a distance-dependent interactionwith the acceptor label, which interaction produces a detectable signal.

In this assay, aggregation of the membrane protein is detected by virtueof the inability of the donor labels and acceptor labels to label thetermini of the membrane protein when aggregated. When the membraneprotein aggregates the labelling sites on the membrane protein becomeinaccessible and so the donor and acceptor labels cannot come intoproximity with each other, and thereby cannot generate a detectablesignal. If the membrane proteins are stable, there is less aggregationupon exposure to a destabilising agent and/or condition and so moresignal, whereas if the membrane proteins are unstable, there is moreaggregation upon exposure to a destabilising agent and/or condition andso less signal.

Preferences for the membrane protein population, donor label andacceptor label include those described above in relation to the firstaspect of the invention.

Conveniently, one of the N-terminus or C-terminus of the membraneprotein is capable of being labelled non-covalently with a donor label,and the other of the N-terminus or C-terminus of the membrane protein iscapable of being labelled non-covalently with an acceptor label. Thus,the membrane protein may be engineered to contain a moiety at the N-and/or C-terminus that is capable of, either directly or indirectly,binding to the donor or acceptor label non-covalently. For example, theN-terminus and/or C-terminus may contain a tag, such as a c-Myc tag,recognised by an antibody that, in turn, is labelled with the donorlabel or acceptor label. Similarly, the N-terminus and/or C-terminus maycontain a moiety that is recognised by one member of a binding partnerpair (eg biotin and streptavidin/avidin), the other member of thebinding partner pair, in turn, being labelled with the donor label oracceptor label. Example 1 illustrates this principle whereby a GPCR isN-terminally tagged with a biotin acceptor peptide (BAP) and isC-terminally tagged with a c-Myc tag. The GPCR may then be labelled bymixing with streptavidin labelled with d2 and anti-c-Myc antibodylabelled with terbium. By labelling at the termini, the labels do notneed to be capable of recognising the tertiary structure of the membraneprotein, and so a loss of labelling is a direct measure of proteinaggregation that is not skewed by changes in ligand or antibody bindingsites that are not necessarily correlated with aggregation.

It is appreciated that one of the termini be labelled prior to step (b),provided that the labelling can withstand the stability modulating agentand/or condition.

Preferably the denaturant/denaturing condition is removed beforelabelling of one or both of the termini, so as to minimise adverselyaffecting the labelling procedure, as described further below.

Following exposure to the stability modulating agent and/or condition,the termini of the membrane protein are labelled. Either the N-terminusis labelled with a donor label and the C-terminus with an acceptorlabel, or the C-terminus is labelled with a donor label and theN-terminus with a donor label.

It is preferred if the amount of label that is added to the membraneprotein is an amount that gives the maximal distance-dependentinteraction signal while using the least amount of labelling reagents.The amount can be readily determined empirically and will generallydepend on the choice of label and expression levels of membrane protein.Optimising the amount may involve doing a titration matrix of varyingamounts of acceptor and donor labels, on a lysate containing themembrane protein of interest as well as a mock lysate. The optimalamount is one that gives the greatest difference in distance-dependentinteraction signal (eg FRET signal) as between the lysate containing themembrane protein, and the mock lysate, while using the least amount oflabelling reagents. The inventors have performed such an optimisationfor streptavidin-d2 and Myc-Tb, and identified respective optimumconcentrations of 1 nM and 100 nM.

Following activation of the donor label to permit a distance-dependentinteraction with the acceptor label, the level of aggregation is thenassessed.

In an embodiment, it may be desirable to also perform steps (a) and (c)of the method without performing step (b). This would provide anindication of background aggregation when the membrane protein is notexposed to a stability modulating agent and/or condition that can becompared to the level of aggregation when the membrane protein isexposed to a stability modulating agent and/or condition. In otherwords, the assay may be preceded by a step wherein the aggregationbetween membrane proteins of the first and second populations isassessed without exposure to a stability modulating agent and/orcondition.

It will be appreciated that the invention also provides a membraneprotein (eg GPCR) wherein one of the N-terminus or C-terminus isattached to a donor label (eg donor fluorophore) and the other of theN-terminus or C-terminus is attached to an acceptor label (eg acceptorfluorophore). Thus, any labelled membrane protein (eg GPCR) that is usedin the assay of the second aspect of the invention is included in thescope of the invention. Preferences for the membrane proteins, donorlabel and acceptor label are as defined above. Thus, the membraneprotein may be a GPCR that has the donor fluorophore (eg terbium)attached to the N-terminus and a donor fluorophore (eg EGFP) attached tothe C-terminus. It will be understood that the attachment of the labelsto the termini of the membrane protein need not be direct, but may beindirect, eg by virtue of the label being attached to a further moiety,such as a terminal tag, which in turn is attached to the membraneprotein. It will also be understood that the labels may be attachednon-covalently or covalently to the termini of the membrane protein. Theinvention also provides the use of such a membrane protein in an assayfor measuring the conformational stability of the membrane protein.

The stability modulating agent and/or condition in the assays of theinvention is preferably a denaturing agent and/or condition, for exampleone selected from one or more of heat, pH, a detergent, or a chaotropicagent. It will be appreciated that any denaturing agent and/or conditionmay be used which is known to modulate the secondary and tertiarystructure of a protein but not the primary structure of a protein.

In relation to stability to heat (ie thermostability), it may beconvenient to determine a “quasi T_(m)” or “quasi T_(agg)” ie thetemperature at which 50% of the membrane protein (eg GPCR) is aggregatedunder stated conditions after incubation for a given period of time (eg30 minutes). Mutant membrane proteins that have higher thermostabilityhave an increased quasi Tm or Tagg compared to their parents.

In relation to stability to a detergent or to a chaotrope, typically themembrane protein is incubated for a defined time in the presence of atest detergent or a test chaotropic agent and the stability isdetermined using an assay of the invention.

In relation to an extreme of pH, a typical test pH would be chosen (egin the range 4.5 to 5.5 (low pH) or in the range 8.5 to 9.5 (high pH).

Because relatively harsh detergents are used during crystallisationprocedures, it is preferred that membrane proteins (eg GPCRs) are stablein the presence of such detergents. The order of “harshness” of certaindetergents is DDM, C₁₁→C₁₀→C₉→C₈ maltoside or glucoside,lauryldimethylamine oxide (LDAO) and SDS. It is particularly preferredif the membrane protein (eg GPCR) is more stable to any of C₉ maltosideor glucoside, C₈ maltoside or glucoside, LDAO and SDS, and so it ispreferred that these detergents are used for stability testing.

It will be appreciated that heat is acting as the destabilisingcondition, and this can readily be removed by cooling the sample, forexample by placing on ice. It is believed that thermostability may alsobe a guide to the stability to other denaturing or destabilising agentsand/or conditions. Thus, increased thermostability is likely totranslate into stability in destabilising detergents, especially thosethat are more destabilising than DDM, eg those detergents with a smallerhead group and a shorter alkyl chain and/or with a charged head group.We have found that a thermostable GPCR is also more stable towards harshdetergents, for example.

When an extreme of pH is used as the destabilising condition, it will beappreciated that this can be removed quickly by adding a neutralisingagent. Similarly, when a chaotrope is used as a destabilizing agent, thedestabilising effect can be removed by diluting the sample below theconcentration in which the chaotrope exerts its chaotropic effect.

A considerable advantage of the assays of the invention is that they donot need to include a separation step to remove labelled ligand. This isan important difference from known methods of measuring membrane proteinstability which involve use of a radiolabelled ligand. In such knownmethods, unbound radiolabel must be removed in a washing step prior todetection of the radiolabel. This separation step makes the knownmethods laborious and unsuitable for high throughput applications.

Although the assays of the invention do not use ligand binding as anindicator of the stability of a membrane protein, it may be beneficialto include a ligand of the membrane protein in the sample provided instep (a). The presence of a ligand, especially when the membrane proteinis a GPCR, can improve stability and/or increase the probability ofstabilising the GPCR in a desired conformation. Similarly, particularconformational states may be enriched for by varying physiochemicalparameters or using additives such as any of pH, salt, metal ions,temperature, chaotropic agents, and glycerol. One may optimisephysiochemical parameters to favour a particular conformation by doingstandard pharmacological characterisations such as ligand bindingaffinities or functional assays under different parameters.

By “ligand” we include any molecule which binds to a membrane proteinsuch as a GPCR.

When the membrane protein is a GPCR, the ligand will typically bind toone conformation of the GPCR (and may cause the GPCR to adopt thisconformation), but does not bind as strongly to another conformationthat the GPCR may be able to adopt. Thus, the presence of the ligand maybe considered to encourage the GPCR to adopt the particularconformation. Preferably the ligand is from the agonist class of ligandsand the particular conformation is an agonist conformation, or theligand is from the antagonist class of ligands and the particularconformation is an antagonist conformation.

Many suitable ligands are known.

Typically, the ligand is a full agonist and is able to bind to themembrane protein (eg a receptor such as a GPCR) and is capable ofeliciting a full (100%) biological response, measured for example byG-protein coupling, downstream signalling events or a physiologicaloutput such as vasodilation. Thus, typically, the biological response isGDP/GTP exchange in a G-protein, followed by stimulation of the linkedeffector pathway. The measurement, typically, is GDP/GTP exchange or achange in the level of the end product of the pathway (eg cAMP, cGMP orinositol phosphates). The ligand may also be a partial agonist and isable to bind to the GPCR and is capable of eliciting a partial (<100%)biological response.

The ligand may also be an inverse agonist, which is a molecule whichbinds to a membrane protein (eg a receptor such as a GPCR) and reducesits basal (ie unstimulated by agonist) activity sometimes even to zero.

The ligand may also be an antagonist, which is a molecule which binds toa membrane protein (eg a receptor such as a GPCR) and blocks binding ofan agonist, so preventing a biological response. Inverse agonists andpartial agonists may under certain assay conditions be antagonists.

The above ligands may be orthosteric, by which we include the meaningthat they combine with the same site as the endogenous agonist; or theymay be allosteric or allotopic, by which we include the meaning thatthey combine with a site distinct from the orthosteric site. The aboveligands may be syntopic, by which we include the meaning that theyinteract with other ligand(s) at the same or an overlapping site. Theymay be reversible or irreversible.

In relation to antagonists, they may be surmountable, by which weinclude the meaning that the maximum effect of agonist is not reduced byeither pre-treatment or simultaneous treatment with antagonist; or theymay be insurmountable, by which we include the meaning that the maximumeffect of agonist is reduced by either pre-treatment or simultaneoustreatment with antagonist; or they may be neutral, by which we includethe meaning the antagonist is one without inverse agonist or partialagonist activity. Antagonists typically are also inverse agonists.

Ligands for use in the invention may also be allosteric modulators suchas positive allosteric modulators, potentiators, negative allostericmodulators and inhibitors. They may have activity as agonists or inverseagonists in their own right or they may only have activity in thepresence of an agonist or inverse agonist in which case they are used incombination with such molecules in order to bind to the membrane protein(eg GPCR).

Neubig et al (2003) Pharmacol. Rev. 55, 597-606, incorporated herein byreference, describes various classes of ligands.

Preferably, the above-mentioned ligands are small organic or inorganicmoieties, but they may be peptides or polypeptides. Typically, when theligand is a small organic or inorganic moiety, it has a M_(r) of from 50to 2000, such as from 100 to 1000, for example from 100 to 500.

Typically, the ligand binds to the membrane protein (eg GPCR) with aK_(d) of from mM to pM, such as in the range of from μM (micromolar) tonM. Generally, the ligands with the lowest Kd are preferred.

Small organic molecule ligands are well known in the art. Other smallmolecule ligands include 5HT which is a full agonist at the 5HT1Areceptor; eltoprazine which is a partial agonist at the 5HT1A receptor(see Newman-Tancredi et al (1997) Neurophamacology 36, 451-459);(+)-butaclamol and spiperone are dopamine D2 receptor inverse agonists(see Roberts & Strange (2005) Br. J. Pharmacol. 145, 34-42); andWIN55212-3 is a neutral antagonist of CB2 (Savinainen et al (2005) Br.J. Pharmacol. 145, 636-645).

The ligand may be a peptidomimetic, a nucleic acid, a peptide nucleicacid (PNA) or an aptamer. It may be an ion such as Na⁺ or Zn²⁺, a lipidsuch as oleamide, or a carbohydrate such as heparin.

The ligand may be a polypeptide which binds to the membrane protein (egreceptor such as a GPCR). Such polypeptides (by which we includeoligopeptides) are typically from M_(r) 500 to M_(r) 50,000, but may belarger. The polypeptide may be a naturally occurring GPCR-interactingprotein or other protein which interacts with the GPCR, or a derivativeor fragment thereof, provided that it binds selectively to the GPCR in aparticular conformation. GPCR-interacting proteins include thoseassociated with signalling and those associated with trafficking, whichoften act via PDZ domains in the C terminal portion of the GPCR.

Polypeptides which are known to bind certain GPCRs include any of a Gprotein, an arrestin, a RGS protein, G protein receptor kinase, a RAMP,a 14-3-3 protein, a NSF, a periplakin, a spinophilin, a GPCR kinase, areceptor tyrosine kinase, an ion channel or subunit thereof, an ankyrinand a Shanks or Homer protein. Other polypeptides include NMDA receptorsubunits NR1 or NR2a, calcyon, or a fibronectin domain framework. Thepolypeptide may be one which binds to an extracellular domain of a GPCR,such as fibulin-1. The polypeptide may be another GPCR, which binds tothe selected GPCR in a hetero-oligomer. A review of protein-proteininteractions at GPCRs is found in Milligan & White (2001) TrendsPharmacol. Sci. 22, 513-518, or in Bockaert et al (2004) Curr. OpinionDrug Discov. Dev. 7, 649-657 incorporated herein by reference.

The polypeptide ligand may conveniently be an antibody which binds tothe membrane protein (eg GPCR). By the term “antibody” we includenaturally-occurring antibodies, monoclonal antibodies and fragmentsthereof. We also include engineered antibodies and molecules which areantibody-like in their binding characteristics, including single chainFv (scFv) molecules and domain antibodies (dAbs). Mention is also madeof camelid antibodies and engineered camelid antibodies. Such moleculeswhich bind GPCRs are known in the art and in any event can be made usingwell known technology. Suitable antibodies include ones presently usedin radioimmunoassay (RIAs) for GPCRs since they tend to recogniseconformational epitopes.

The polypeptide may also be a binding protein based on a modularframework, such as ankyrin repeat proteins, armadillo repeat proteins,leucine rich proteins, tetratriopeptide repeat proteins or DesignedAnkyrin Repeat Proteins (DARPins) or proteins based on lipocalin orfibronectin domains or Affilin scaffolds based on either human gammacrystalline or human ubiquitin.

It will be appreciated that the use of antibodies, or other “universal”binding polypeptides (such as G-proteins which are known to couple withmany different GPCRs) may be particularly advantageous in the use of themethod on “orphan” GPCRs for which the natural ligand, and smallmolecule ligands, are not known.

It will be appreciated that the above assays may be used in methods toselect membrane proteins (eg GPCRs) with increased conformationalstability. Such methods have the advantage over existing methods thatthere is no need for a separation step to remove labelled ligand. Also,the method can be performed even where a good radioligand with highaffinity and/or favourable properties in detergent is not available.

Accordingly, a third aspect of the invention provides a method forselecting a membrane protein (eg GPCR) with increased conformationalstability, comprising:

(a) comparing the conformational stability of one or more mutants of aparent membrane proteins with the conformational stability of the parentmembrane protein according to the assay of the first or second aspectsof the invention, and

(b) selecting one or more mutants that have increased conformationalstability relative to the parent membrane protein.

By including a ligand in either assay of the invention as describedabove, the method of the third aspect of the invention may be consideredto be a way of selecting membrane proteins (eg GPCRs) which are trappedin a conformation of biological relevance (eg ligand bound state), andwhich are more stable with respect to that conformation. Preferably, theligand is from the agonist class of ligands and the particularconformation is an agonist conformation, or the ligand is from theantagonist class of ligands and the particular conformation is anantagonist conformation.

The mutant membrane proteins (eg GPCRs) may be prepared by any suitablemethod. Conveniently, the mutant protein is encoded by a suitablenucleic acid molecule and is expressed in a suitable host cell. Thepreparation of suitable nucleic acid molecules, expression systems andhost cells include those described above.

In a particular embodiment of the invention, the membrane protein isCCR5, the donor label is terbium and the acceptor label is EGFP. Terbiummay be attached to the N-terminus by using SNAP tag technology. EGFP maybe attached to the C-terminus by being expressed as a C-terminal fusion.

In a further preferred embodiment of the invention, the membrane proteinis CCR5, the donor label is terbium and the acceptor label is d2.Terbium may be non-covalently attached to the C-terminus via labellingan anti-c-Myc antibody that is bound to a c-Myc epitope at theC-terminus. d2 may be non-covalently attached to the N-terminus vialabelling streptavidin that is bound to biotin which in turn bindsbiotin acceptor peptide (BAP) fused to the N-terminus.

In a particular embodiment of the invention, the membrane protein is aGPCR such as any of GLP1R, CMKLR, or TGR5 (GPBAR-I), the donor label isterbium and the acceptor label is EGFP. Terbium may be attached to theN-terminus by using SNAP tag technology. EGFP may be attached to theC-terminus by being expressed as a C-terminal fusion.

In a further preferred embodiment of the invention, the membrane proteinis a GPCR such as any of GLP1R, CMKLR, or TGR5 (GPBAR-I), the donorlabel is terbium and the acceptor label is d2. Terbium may benon-covalently attached to the C-terminus via labelling an anti-c-Mycantibody that is bound to a c-Myc epitope at the C-terminus. d2 may benon-covalently attached to the N-terminus via labelling streptavidinthat is bound to biotin which in turn binds biotin acceptor peptide(BAP) fused to the N-terminus.

The invention will now be described with the aid of the followingFigures and Examples:

FIG. 1: (a) Principle of the intermolecular aggregation assay. Terbiumlabelled or EGFP-tagged receptors are solubilised and purified. The 1:1mixed samples are heated and FRET is measured. (b) An example ofreceptor stabilities measured using this assay, the values denote Tagg.

FIG. 2: (a) Principle of the intramolecular aggregation assay. In vivobiotinylated receptors are solubilised and incubated at differenttemperatures. The lysates are then mixed with d2 labelled streptavidinand terbium labelled anti-cMyc antibody and FRET is measured. Upon heatinduced aggregation, the levels of FRET are reduced due to theinaccessibility of the sites to label the receptor. (b) An example ofreceptor stabilities measured using this assay, the values denote Tagg.U represents the FRET values measured in lysates prepared from mocktransfected cells. FRET ratio represents emission at 650 nm normalisedto the emission at 620 nm.

FIG. 3: Stability of wild-type and mutant (T355F) GLP1R measured usingintramolecular aggregation assay. Wild-type and T355F GLP1R constructs(both N-terminally BAP tagged and C-terminally c-Myc tagged) wereco-expressed in HEK293T cells with BirA. Following solubilisation in 1%DDM, samples were incubated at different temperatures for 30 minutes.FRET was measured after addition of d2 labelled streptavidin and terbiumlabelled anti-cMyc antibody.

EXAMPLE 1: METHODOLOGY FOR RECEPTOR STABILISATION

Our conventional method for receptor stabilisation uses ligand bindingto measure the levels of active receptor. Briefly, solubilised receptorsthat have been incubated with a radio-labelled ligand are heated atdifferent temperatures for a set amount of time. Next, the excess andunbound ligand is separated from the receptor bound ligand and theresidual level of radioactivity is measured. Plotting this data againsttemperature gives a thermal decay curve and the Tm value is defined asthe temperature at which 50% of receptor activity is retained. Obviouslythis method relies on the availability of a good radioligand with highaffinity and favourable properties in detergent. There has thereforebeen a need to develop new methodologies that would allow us tostabilise receptors in cases where such a ligand is not available. Werefer to these methodologies as ligand-independent methods which meansthat ligand binding ability of the receptor is not used to measurereceptor activity, although, the ligand can be present to increasestability or increase the probability of stabilising the receptor in thedesired conformation.

Intermolecular Aggregation Assay

Protein aggregation upon unfolding is a general feature of all proteinsthat occurs to varying degrees in different proteins. Membrane proteinsare known to exhibit high levels of aggregation after solubilisation andmore so after denaturation. It is therefore possible to use the levelsof aggregation as a measure of global stability.

An assay that allows aggregation to be measured in an easy andminiaturisable format would be a useful tool to generate a stablereceptor. There are a number of different ways that aggregation could bemeasured including the biophysical methods described above. Most ofthese methods require high amounts of very pure protein.

We have developed a method that allows receptor aggregation to bemeasured from small amounts of semi-purified preparations. In order tomeasure the receptor aggregation using this assay, two populations ofthe receptor are labelled with either a FRET acceptor group or a FRETdonor group (FIG. 1a ). We have used the SNAP tag technology to labelthe receptors N-terminally with terbium that acts as the FRET donor. TheFRET acceptor is EGFP that is expressed as a C-terminal fusion. Thesetwo populations of the receptor are expressed, solubilised and partiallypurified separately. The semi-pure preps of the two sets are mixed in a1:1 ratio and the mixture is incubated at different temperatures for aset amount of time. As the receptors unfold and aggregates are formed,FRET acceptor and donor are brought in close proximity that results inFRET emission. Increase in FRET is plotted against temperature and thetemperature at which 50% FRET is observed is defined as T_(agg) (FIG. 1b).

Intramolecular Aggregation Assay

This assay is a variation on the previous method. In this assay, theFRET acceptor and donor are placed on the N- and C-termini of the samereceptor molecule. However, importantly, the receptor is solubilised andheated prior to labelling with the FRET acceptor and donor. So, as thereceptor unfolds and aggregates the sites of FRET acceptor and donorlabelling are obscured and thus become inaccessible. This in turnresults in loss of FRET as a function of temperature and the T_(agg) isdefined as the temperature at which 50% FRET is observed (FIG. 2a ). Inthis example, the receptor is tagged N-terminally with a biotin acceptorpeptide (BAP) and is C-terminally tagged with cMyc tag. The receptor isco-expressed with biotin ligase A (BirA) enzyme which leads to theexpression of an N-terminally biotinylated receptor. Followingsolubilisation, the crude lysate is incubated at different temperaturesfor a set amount of time. The lysates are then mixed with streptavidinlabelled with d2 and anti-cMyc antibody labelled with terbium. Thelevels of FRET are then measured and plotted against temperature (FIG.2b ).

In order to see if this method was capable of identifyingthermostabilisng mutations, a number of mutants were generated in theplasmid encoding GLP1R using site directed mutagenesis. Theintramolecular aggregation assay was then used to assess the thermalstability of the mutants compared to wild-type GLP1R. FIG. 3 showsexemplary data for T355F mutation that confers ˜6° C. increase inthermal stability of GLP1R. As seen from the figure, the value of Taggfor the stabilised mutant GLP1R is higher than that of the wild-typeGLP1R.

Materials and Methods

Receptor Expression

In all cases described, the receptors were expressed transiently inHEK293T cells. Briefly, cells were seeded at the density of 3×10⁶ cellsin 10 cm petri dishes containing Dulbecco's Modified Eagle Medium (DMEM)supplemented with 10% foetal bovine serum (FBS) and incubated overnightin 37° C. incubator. The next day, cells were transfected using 6 ug ofplasmid encoding the receptor of interest using GeneJuice according tothe manufacturer's instructions. Cells were incubated for about 40 hourspost-transfection at the 37° C. incubator.

Receptor Solubilisation

Following transfection cells were harvested in phosphate buffered salineand washed once in the same buffer. Cells were then solubilised in total1 mL of solubilisation buffer containing 50 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 150 mMsodium chloride adjusted to pH 7.5, supplemented with 1%n-Dodecyl-R-maltoside (DDM) and protease inhibitor cocktail. Receptorswere solubilised for 1 hour with end-to-end rotation. All solubilisationsteps as well as the subsequent steps were carried out at 4° C.

Intermolecular Aggregation Assay

SNAP-tagged and EGFP-tagged receptors were expressed separately inHEK293T cells as described above. Prior to solubilisation, SNAP-taggedreceptors were harvested and re-suspended in DMEM plus FBS containing250 nM of SNAP-Lumi4Tb (Cisbio) and incubated for 1 hour at 37° C.incubator to label the N-terminus. Cells were then washed three timeswith 1 mL of PBS to remove the excess unbound SNAP-Lumi4Tb. After thelast wash cells were solubilised in parallel along with cells expressingEGFP tagged receptor in 1% DDM. The crude lysates were clarified bycentrifugation at 13000 rpm for 10 minutes at 4° C. The cleared lysateswere incubated with 250 uL of 50% slurry Ni-NTA agarose resinpre-equilibrated in the solubilisation buffer in order to purifyreceptors using the C-terminal 10× His tag. The mixture was incubated at4° C. with end-to-end rotation for 1.5 hour and then washed 3× with 1 mLof chilled wash buffer containing 50 mM HEPES, 150 mM NaCl, 0.03% DDM,20 mM Imidazole, pH adjusted to 7.5. After the last wash receptors wereeluted in 500 uL of elution buffer containing 50 mM HEPES, 150 mM NaCl,0.03% DDM and 100 mM Histidine, pH adjusted to 7.5. The Lumi4Tb taggedand EGFP tagged samples were mixed 1:1 and aliquots were incubated atincreasing temperatures for 30 minutes. The samples were then returnedto 4° C. and transferred to white 96-well plates and FRET was measuredbetween Lumi4Tb and EGFP using PHERAstar Plus (BMG Labtech) instrument.The FRET settings were according to the recommendations of themanufacturer.

Intramolecular Aggregation Assay

Receptors tagged N-terminally with the biotin acceptor tag (BAP) andC-terminally with c-Myc tag were expressed transiently in HEK293T cellsas described above. It is notable that the plasmid also encodes for theBirA enzyme that mediates biotinylation on the BAP tag. Cells wereincubated with 100 uM of biotin during expression. Following harvestingand solubilisation, aliquots of cleared lysates were incubated atincreasing temperatures for 30 minutes. Samples were cooled to 4° C. and20 uL of each sample was added to 20 uL of FRET mixture pre-aliquoted inwhite 384 well plate containing 2 nM anti-cMyc antibody conjugated toterbium (Cisbio) and 200 nM Streptavidin conjugated to d2 fluorophore(Cisbio). The plate was incubated overnight at 4° C. before measuringthe FRET signal on PHERAstar Plus according to the recommendations ofthe manufacturer.

TABLE A Official IUPHAR Human gene Rat gene Mouse gene name name namename 5HT1a HTR1A Htr1a Htr1a 5HT2A HTR2A Htr2a Htr2a 5HT2C HTR2C Htr2cHtr2c 5HT6 HTR6 Htr6 Htr6 5HT7 HTR7 Htr7 Htr7 M1 CHRM1 Chrm1 Chrm1 M2CHRM2 Chrm2 Chrm2 M3 CHRM3 Chrm3 Chrm3 M4 CHRM4 Chrm4 Chrm4 M5 CHRM5Chrm5 Chrm5 C3a C3AR1 C3ar1 C3ar1 C5a C5R1 C5r1 C5r1 C5L2 GPR77 Gpr77Gpr77 AT1 AGTR1 Agtr1b Agtr1b APJ AGTRL1 Agtrl1 Agtrl1 GPBA GPBAR1Gpbar1 Gpbar1 BB1 NMBR Nmbr Nmbr BB2 GRPR Grpr Grpr BB3 BRS3 Brs3 Brs3BK1 BDKRB1 Bdkrb1 Bdkrb1 BK2 BDKRB2 Bdkrb2 Bdkrb2 CB1 CNR1 Cnr1 Cnr1 CB2CNR2 Cnr2 Cnr2 CCR1 CCR1 Ccr1 Ccr1 CCR2 CCR2 Ccr2 Ccr2 CCR3 CCR3 Ccr3Ccr3 CCR4 CCR4 Ccr4 Ccr4 CCR5 CCR5 Ccr5 Ccr5 CCR6 CCR6 Ccr6 Ccr6 CCR7CCR7 Ccr7 Ccr7 CCR8 CCR8 Ccr8 Ccr8 CCR9 CCR9 Ccr9 Ccr9 CCR10 CCR10 Gpr2Gpr2 CXCR1 IL8RA Il8ra Il8ra CXCR2 IL8RB Il8rb Il8rb CXCR3 CXCR3 Cxcr3Cxcr3 CXCR4 CXCR4 Cxcr4 Cxcr4 CXCR5 CXCR5 Blr1 Blr1 CXCR6 CXCR6 Cxcr6Cxcr6 CX3CR1 CX3CR1 Cx3cr1 Cx3cr1 XCR1 XCR1 Xcr1 Xcr1 DRD1 DRD1 Drd1aDrd1a DRD2 DRD2 Drd2 Drd2 DRD3 DRD3 Drd3 Drd3 DRD4 DRD4 Drd4 Drd4 DRD5DRD5 Drd5 Drd5 GPER GPER Gpr30 Gper FPR1 FPR1 Fpr1 Fpr1 FPR2/ALX FPR2Fpr2 Fpr2 FPR3 FPR3 Fpr3 Fpr3 FFA1 FFAR1 Ffar1 Ffar1 FFA2 FFAR2 Gpr43Ffar2 FFA3 FFAR3 Ffar3 Ffar3 GALR1 GALR1 Galr1 Galr1 GALR2 GALR2 Galr2Galr2 GALR3 GALR3 Galr3 Galr3 ghrelin GHSR Ghsr Ghsr FSH FSHR Fshr FshrLH LHCGR Lhcgr Lhcgr GnRH GNRHR Gnrhr Gnrhr GnRH2 GNRHR2 KiSS1 KISS1RKiss1r Kiss1r OXE OXER1 FPR2/ALX FPR2 Fpr2 Fpr2 LPAR1 LPAR1 Lpar1 Lpar1LPAR2 LPAR2 Lpar2 Lpar2 LPAR3 LPAR3 Lpar3 Lpar3 S1PR1 S1PR1 S1pr1 S1pr1S1PR2 S1PR2 S1pr2 S1pr2 S1PR3 S1PR3 S1pr3 S1pr3 S1PR4 S1PR4 S1pr4 S1pr4S1PR5 S1PR5 S1pr5 S1pr5 MCHR1 MCHR1 Mchr1 Mchr1 MCHR2 MCHR2 MC1R MC1RMc1r Mc1r MC2R MC2R Mc2r Mc2r MC3R MC3R Mc3r Mc3r MC4R MC4R Mc4r Mc4rMC5R MC5R Mc5r Mc5r MTNR1A MTNR1A Mtnr1a Mtnr1a MTNR1B MTNR1B Mtnr1bMtnr1b NMU1 NMUR1 Nmur1 Nmur1 NMU2 NMUR2 Nmur2 Nmur2 NPFF1 NPFFR1 Npffr1NPFF2 NPFFR2 Npffr2 Npffr2 NPS NPSR1 Npsr1 Npsr1 NPBW1 NPBWR1 Npbwr1Npbwr1 NPBW2 NPBWR2 NPY1 NPY1R Npy1r Npy1r NPY2R NPY2R Npy2r Npy2r PPYR1PPYR1 Ppyr1 Ppyr1 NPY5R NPY5R Npy5r Npy5r NTSR1 NTSR1 Ntsr Ntsr NTSR2NTSR2 Ntsr2 Ntsr2 GPR81 GPR81 Gpr81 Gpr81 (temporary name) GPR109AGPR109A Gpr109a Gpr109a (temporary name) GPR109B GPR109B (temporaryname) Delta OPRD1 Oprd1 Oprd1 Kappa OPRK1 Oprk1 Oprk1 Mu OPRM1 Oprm1Oprm1 NOP OPRL1 Oprl Oprl1 OX1 HCRTR1 Hcrtr1 Hcrtr1 OX2 HCRTR2 Hcrtr2Hcrtr2 P2RY2 P2RY2 P2ry2 P2ry2 P2RY11 P2RY11 P2RY12 P2RY12 P2ry12 P2ry12PROKR1 PROKR1 Prokr1 Prokr1 PROKR2 PROKR2 Prokr2 Prokr2 PRRP PRLHR PrlhrPrlhr PAR1 F2R F2r F2r PAR2 F2RL1 F2rl1 F2rl1 RXFP1 RXFP1 Rxfp1 Rxfp1RXFP2 RXFP2 Rxfp2 Rxfp2 RXFP3 RXFP3 Rxfp3 Rxfp3 RXFP4 RXFP4 Rxfp4 SSTR1SSTR1 Sstr1 Sstr1 SSTR2 SSTR2 Sstr2 Sstr2 SSTR5 SSTR5 Sstr5 Sstr5 V1AAVPR1A Avpr1a Avpr1a V1B AVPR1B Avpr1b Avpr1b V2 AVPR2 Avpr2 Avpr2 CCRL2CCRL2 Ccrl2 Ccrl2 CMKLR1 CMKLR1 Cmklr1 Cmklr1 CMKOR1 CMKOR1 Rdc1 Cmkor1CT CALCR Calcr Calcr CALCRL CALCRL Calcrl Calcrl CRF1 CRHR1 Crhr1 Crhr1CRF2 CRHR2 Crhr2 Crhr2 GHRH GHRHR Ghrhr Ghrhr GIP GIPR Gipr Gipr GLP-1GLP1R Glp1r Glp1r GLP-2 GLP2R Glp2r Glp2r glucagon GCGR Gcgr Gcgrsecretin SCTR Sctr Sctr PTH1 PTH1R Pth1r Pth1r PTH2 PTHR2 Pthr2 Pthr2PAC1 ADCYAP1R1 Adcyap1r1 Adcyap1r1 VPAC1 VIPR1 Vipr1 Vipr1 VPAC2 VIPR2Vipr2 Vipr2 CaS CASR Casr Casr GABBR1 GABBR1 Gabbr1 Gabbr1 GABBR2 GABBR2Gabbr2 Gabbr2 mGluR1 GRM1 Grm1 Grm1 mGluR2 GRM2 Grm2 Grm2 mGluR3 GRM3Grm3 Grm3 mGluR4 GRM4 Grm4 Grm4 mGluR5 GRM5 Grm5 Grm5 mGluR6 GRM6 Grm6Grm6 mGluR7 GRM7 Grm7 Grm7 mGluR8 GRM8 Grm8 Grm8

TABLE B Official IUPHAR Human gene Rat gene Mouse gene receptor namename name name CCRL2 CCRL2 Ccrl2 Ccrl2 CMKLR1 CMKLR1 Cmklr1 Cmklr1CMKOR1 CMKOR1 Rdc1 Cmkor1 EBI2 GPR183 Gpr183 Gpr183 GPR1 GPR1 Gpr1 Gpr1GPR3 GPR3 Gpr3 Gpr3 GPR4 GPR4 Gpr4 Gpr4 GPR6 GPR6 Gpr6 Gpr6 GPR12 GPR12Gpcr12 Gpr12 GPR15 GPR15 Gpr15 Gpr15 GPR17 GPR17 Gpr17 Gpr17 GPR18 GPR18Gpr18 Gpr18 GPR19 GPR19 Gpr19 Gpr19 GPR20 GPR20 Gpr20 Gpr20 GPR21 GPR21Gpr21 Gpr21 GPR22 GPR22 Gpr22 Gpr22 GPR23 GPR23 Gpr23_predicted Gpr23GPR25 GPR25 Gpr25 Gpr25 GPR26 GPR26 Gpr26 Gpr26 GPR27 GPR27 Gpr27 Gpr27GPR31 GPR31 Gpr31 Gpr31c GPR32 GPR32 GPR34 GPR34 GPR34 Gpr34 GPR35 GPR35Gpr35 Gpr35 GPR37 GPR37 Gpr37 Gpr37 GPR37L1 GPR37L1 Gpr37l1 Gpr37l1GPR39 GPR39 Gpr39 Gpr39 GPR45 GPR45 Gpr45 Gpr45 GPR50 GPR50 Gpr50 Gpr50GPR52 GPR52 Gpr52 Gpr52 GPR55 GPR55 Gpr55 Gpr55 GPR61 GPR61 Gpr61 Gpr61GPR62 GPR62 RGD1560166 Gpr62 GPR63 GPR63 Gpr63 Gpr63 GPR65 GPR65 Gpr65Gpr65 GPR68 GPR68 Gpr68 Gpr68 GPR75 GPR75 Gpr75 Gpr75 GPR78 GPR78 GPR82GPR82 Gpr82 GPR83 GPR83 Gpr83 Gpr83 GPR84 GPR84 Gpr84 Gpr84 GPR85 GPR85Gpr85 Gpr85 GPR87 GPR87 Gpr87 Gpr87 GPR88 GPR88 Gpr88 Gpr88 GPR92 GPR92RGD1562580_predicted Gpr92 GPR101 GPR101 Gpr101 Gpr101 GPR119 GPR119Gpr119 Gpr119 GPR120 GPR120 Gpr120 Gpr120 GPR132 GPR132 Gpr132 Gpr132GPR135 GPR135 Gpr135 Gpr135 GPR139 GPR139 Gpr139 Gpr139 GPR141 GPR141Gpr141 Gpr141 GPR142 GPR142 Gpr142 Gpr142 GPR146 GPR146 Gpr146 Gpr146GPR148 GPR148 GPR149 GPR149 Gpr149 Gpr149 GPR150 GPR150 Gpr150 Gpr150GPR151 GPR151 Gpr151 Gpr151 GPR152 GPR152 Gpr152 Gpr152 GPR153 GPR153Gpr153 Gpr153 GPR160 GPR160 Gpr160 Gpr160 GPR161 GPR161 RGD1563245Gpr161 GPR162 GPR162 Gpr162 Gpr162 GPR171 GPR171 Gpr171 Gpr171 GPR173GPR173 Gpr173 Gpr173 GPR174 GPR174 Gpr174 Gpr174 GPR182 GPR182 Gpr182Gpr182 LGR4 LGR4 Lgr4 Lgr4 LGR5 LGR5 Lgr5 Lgr5 LGR6 LGR6 Lgr6 Lgr6 MAS1MAS1 Mas1 Mas1 MAS1L MAS1L MRGPRD MRGPRD Mrgprd Mrgprd MRGPRE MRGPREMrgpre Mrgpre MRGPRF MRGPRF Mrgprf Mrgprf MRGPRG MRGPRG Mrgprg MrgprgMRGPRX1 MRGPRX1 Mrgprx1 Mrgprx1 MRGPRX2 MRGPRX2 Mrgprx2 Mrgprx2 MRGPRX3MRGPRX3 Mrga10 Mrgpra9 MRGPRX4 MRGPRX4 OPN3 OPN3 Opn3 Opn3 OPN5 OPN5Opn5 Opn5 OXGR1 OXGR1 Oxgr1 Oxgr1 P2RY5 P2RY5 P2ry5 P2y5 P2RY8 P2RY8P2RY10 P2RY10 P2ry10 P2ry10 SUCNR1 SUCNR1 Sucnr1 Sucnr1 TAAR2 TAAR2Taar2 Taar2 TAAR5 TAAR5 Taar5 Taar5 TAAR6 TAAR6 Taar6 Taar6 TAAR8 TAAR8Taar8a Taar8b TAAR9 TAAR9 Taar9 Taar9 BAI1 BAI1 Bai1 Bai1 BAI2 BAI2 Bai2Bai2 BAI3 BAI3 Bai3 Bai3 CD97 CD97 cd97 Cd97 CELSR1 CELSR1 Celsr1 Celsr1CELSR2 CELSR2 Celsr2 Celsr2 CELSR3 CELSR3 Celsr3 Celsr3 ELTD1 ELTD1Eltd1 Eltd1 EMR1 EMR1 Emr1 Emr1 EMR2 EMR2 EMR3 EMR3 GPR56 GPR56 Gpr56Gpr56 GPR64 GPR64 Gpr64 Gpr64 GPR97 GPR97 Gpr97 Gpr97 GPR98 GPR98 Gpr98Gpr98 GPR110 GPR110 Gpr110 Gpr110 GPR111 GPR111 Gpr111 GPR112 GPR112Gpr112 Gpr112 GPR113 GPR113 Gpr113 Gpr113 GPR114 GPR114 Gpr114 Gpr114GPR115 GPR115 Gpr115 Gpr115 GPR116 GPR116 Gpr116 Gpr116 GPR123 GPR123Gpr123 Gpr123 GPR124 GPR124 Gpr124 Gpr124 GPR125 GPR125 Gpr125 Gpr125GPR126 GPR126 Gpr126 Gpr126 GPR128 GPR128 Gpr128 Gpr128 GPR133 GPR133Gpr133 GPR143 GPR143 Gpr143 Gpr143 GPR144 GPR144 Gpr144 GPR157 GPR157Gpr157 Gpr157 LPHN1 LPHN1 Lphn1 Lphn1 LPHN2 LPHN2 Lphn2 Lphn2 LPHN3LPHN3 Lphn3 Lphn3 GPR156 GPR156 GPR158 GPR158 GPR179 GPR179 RAIG1 GPRC5ARAIG2 GPRC5B RAIG3 GPRC5C RAIG4 GPRC5D

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The invention claimed is:
 1. An assay for assessing the conformationalstability of a membrane protein, comprising: (a) providing a samplecomprising a first population and a second population of a membraneprotein; wherein the membrane protein in the first population islabelled with a donor label and the membrane protein in the secondpopulation is labelled with an acceptor label, or the membrane proteinin the first population is labelled with an acceptor label and themembrane protein in the second population is labelled with a donorlabel, and wherein the populations of membrane protein are provided in asolubilized form, (b) exposing the first and second populations of themembrane protein provided in the solubilized form to a denaturant ordenaturing condition, and (c) assessing aggregation between membraneproteins of the first and second populations by activating the donorlabel to permit a distance-dependent interaction with the acceptorlabel, which interaction produces a detectable signal.
 2. The assay ofclaim 1, wherein the first population and second population of themembrane protein are present in the sample in a 1:1 ratio.
 3. The assayof claim 1, wherein the donor label is covalently attached to themembrane protein and the acceptor label is covalently attached to themembrane protein.
 4. An assay for assessing the conformational stabilityof a membrane protein, comprising: (a) providing a sample comprising amembrane protein population, wherein the population of membrane proteinis provided in a solubilized form, (b) exposing the membrane proteinpopulation to a denaturant or denaturing condition, (c) labelling one ofthe N-terminus or C-terminus of the membrane protein with a donor labeland the other of the N-terminus or C-terminus of the membrane proteinwith an acceptor label, and (d) assessing aggregation of the membraneproteins in the population by activating the donor label to permit adistance-dependent interaction with the acceptor label, whichinteraction produces a detectable signal.
 5. The assay of claim 1,wherein the interaction between the donor label and the acceptor labelinvolves the transfer of energy from a donor fluorophore to an acceptorfluorophore.
 6. The assay of claim 5, wherein the donor fluorophore is alanthanide, optionally wherein the lanthanide is Terbium.
 7. The assayof claim 5, wherein the acceptor fluorophore is EGFP or d2.
 8. The assayof claim 1, wherein the interaction between the donor label and theacceptor label is a chemiluminescent reaction, optionally wherein theinteraction between the donor label and the acceptor label involves thegeneration of singlet oxygen molecules that trigger a chemiluminescentreaction.
 9. The assay of claim 1, wherein the donor label and/oracceptor label is directly attached or indirectly attached to themembrane protein.
 10. The assay of claim 1, wherein the sample comprisesone or more detergents selected from the group consisting of DDM,C11-maltoside, C10-maltoside, C9-maltoside, C8-maltoside, C11-glucoside,C10-glucoside, C9-glucoside, C8-glucoside, LDAO, and SDS.
 11. The assayof claim 1, wherein the denaturant or denaturing condition is selectedfrom one or more of heat, a detergent, a chaotropic agent or pH.
 12. Theassay of claim 1, wherein the membrane protein is a GPCR.
 13. The assayof claim 12, wherein the sample provided in step (a) comprises a GPCRligand, the ligand being one that binds to a GPCR when the GPCR isresiding in a particular conformation.
 14. The assay of claim 13,wherein the GPCR ligand is from the agonist class of ligands and theparticular conformation is an agonist conformation, or the GPCR ligandis from the antagonist class of ligands and the particular conformationis an antagonist conformation.
 15. A method for selecting a membraneprotein with increased conformational stability, comprising: (a)comparing the conformational stability of one or more mutants of aparent membrane proteins with the conformational stability of the parentmembrane protein according to the assay of claim 1, and (b) selectingone or more mutants that have increased conformational stabilityrelative to the parent membrane protein.
 16. The method of claim 15,comprising: (a) providing one or more mutants of a parent membraneprotein; (b) assessing the conformational stability of the one or moremutants of the parent membrane protein; (c) assessing the conformationalstability of the parent membrane protein; and (d) selecting one or moremutants of the parent membrane protein that have increasedconformational stability compared to the conformational stability of theparent protein.
 17. The method of claim 15, wherein the membrane proteinhas increased stability to any of heat, a detergent, a chaotropic agentor an extreme of pH.
 18. A method for preparing a mutant GPCR, themethod comprising: (a) carrying out the method of claim 15, (b)identifying the position or positions of the mutated amino acid residueor residues in the mutant membrane protein or membrane proteins whichhas been selected for increased stability, and (c) synthesising a mutantmembrane protein which contains a replacement amino acid at one or moreof the positions identified.
 19. The assay of claim 1, wherein the assayis used in drug screening.
 20. The assay of claim 4, wherein the assayis used in drug screening.